PCR of challenging samples Flashcards
why are there challenging samples?
DNA will not always be optimum quality
Old samples
Environmental conditions
Other DNA/substances contaminating sample
Can all affect amplification by PCR
DNA degradation
During decomposition, DNA templates can become fragmented or chemically modified
This reduces the number of intact target fragments available for PCR
what does DNA degradation lead to?
PCR failure or poor amplification
Enzymatic degradation
Enzymes internal and external to cell can breakdown DNA
Nucleases
Some associated with cell death (internal)
Some in the environment (external) – microorganisms
non enzymatic degradation
Hydrolysis – breaking of a bond using water. In DNA the base-sugar bond is most susceptible to hydrolytic attack leading in the loss of a base
Oxidative reactions – oxygen-derived free radicals can modify bases, blocking PCR
Radiation can cause lesions to DNA
sample storage
Once sample has been taken, the storage conditions can be controlled
Keep sample dry – reduce bacterial growth and limit hydrolytic enzyme activity
Constant low temperature will increase DNA longevity
FTA cards
Room temperature
Apply sample to FTA matric and nucleic acids are captured and stabilised
Successful amplification of blood after 22 years on FTA card
exposure to high temperatures
High temperatures will cause the covalent bonds within the DNA strands to break
A study has found that this begins at 130 degrees centigrade and that complete degradation occurs at 190 degrees centigrade
what can you do about degraded DNA?
Not much
Therefore, important to preserve samples as well as possible
MiniSTRs have been developed which may have more amplification success with degraded DNA
MiniSTRs
Possible to reduce size of amplicon by putting primers closer to STR – advantage because of degraded DNA
low copy number
High sensitivity DNA profiling
Increase the number of cycles >34
Possible to obtain a profile from less than 100pg DNA
Extreme care required
Samples very susceptible to contamination
Interpretation of profiles much more complicated
Caddy report
PCR inhibition
Humic compounds in soil, water and sediments
Tannic acid in leather
Haem in blood
Collagen in bone
Excessive DNA
Heavy metals
Inhibitors can hinder amplification in different ways
By binding to and inactivating template
Affect taq enzyme activity
Applied Biosystems Quantifiler real-time PCR quantification method has an Internal PCR Control that identifies samples that may contain inhibitors
sample purification
Try to remove inhibitors at the extraction stage
Various approaches using magnetic beads, filters, gel
Some substances will co-purify
sample dilution
By diluting the sample, you can decrease the concentration of the inhibitor
However, you will dilute the DNA as well
